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Loss-of-function analysis is one of the major arsenals we have for understanding gene functions in mammalian cells. For analysis of essential genes, the major challenge is to develop simple methodologies for tight and rapid inducible gene inactivation. One approach involves CRISPR-Cas9-mediated disruption of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect. Here we describe the development of a set of Sleeping Beauty transposon-based vectors for expressing auxin-inducible degron (AID)-tagged genes under the regulation of a tetracycline-controlled promoter. The dual transcriptional and degron-mediated post-translational regulation allows rapid and tight silencing of protein expression in mammalian cells. We demonstrated that both non-essential and essential genes could be targeted in human cell lines using a one-step transfection method. Moreover, multiple genes could be simultaneously or sequentially targeted, allowing inducible inactivation of multiple genes. These resources enable highly efficient generation of conditional gene silencing cell lines to facilitate functional studies of essential genes.

Mitotic slippage involves cells exiting mitosis without proper chromosome segregation. Although degradation of cyclin B1 during prolonged mitotic arrest is believed to trigger mitotic slippage, its upstream regulation remains obscure. Whether mitotic slippage is caused by APC/CCDC20 activity that is able to escape spindle-assembly checkpoint (SAC)-mediated inhibition, or is actively promoted by a change in SAC activity remains an outstanding issue. We found that a major culprit for mitotic slippage involves reduction of MAD2 at the kinetochores, resulting in a progressive weakening of SAC during mitotic arrest. A further level of control of the timing of mitotic slippage is through p31comet-mediated suppression of MAD2 activation. The loss of kinetochore MAD2 was dependent on APC/CCDC20, indicating a feedback control of APC/C to SAC during prolonged mitotic arrest. The gradual weakening of SAC during mitotic arrest enables APC/CCDC20 to degrade cyclin B1, cumulating in the cell exiting mitosis by mitotic slippage.

PARP inhibitors have emerged as effective chemotherapeutic agents for BRCA1/BRCA2-deficient cancers. Another DNA damage response protein, ATM, is also increasingly being recognized as a target for synthetic lethality with PARP inhibitors. As ATM functions in both cell cycle arrest and DNA repair after DNA damage, how cells respond to inhibition of ATM and PARP1 is yet to be defined precisely. We found that loss of ATM function, either in an ATM-deficient background or after treatment with ATM inhibitors (KU-60019 or AZD0156), results in spontaneous DNA damage and an increase in PARylation. When PARP1 is also deleted or inhibited with inhibitors (olaparib or veliparib), the massive increase in DNA damage activates the G2 DNA damage checkpoint kinase cascade involving ATR, CHK1/2, and WEE1. Our data indicated that the role of ATM in DNA repair is critical for the synergism with PARP inhibitors. Bypass of the G2 DNA damage checkpoint in the absence of ATM functions occurs only after a delay. The relative insensitivity of PARP1-deficient cells to PARP inhibitors suggested that other PARP isoforms played a relatively minor role in comparison with PARP1 in synergism with ATMi. As deletion of PARP1 also increased sensitivity to ATM inhibitors, trapping of PARP1 on DNA may not be the only mechanism involved in the synergism between PARP1 and ATM inhibition. Collectively, these studies provide a mechanistic foundation for therapies targeting ATM and PARP1.

Summary One of the most intriguing features of cell-cycle control is that, although there are multiple cyclin-dependent kinases (CDKs) in higher eukaryotes, a single CDK is responsible for both G1-S and G2-M in yeasts. By leveraging a rapid conditional silencing system in human cell lines, we confirm that CDK1 assumes the role of G1-S CDK in the absence of CDK2. Unexpectedly, CDK1 deficiency does not prevent mitotic entry. Nonetheless, inadequate phosphorylation of mitotic substrates by noncanonical cyclin B-CDK2 complexes does not allow progression beyond metaphase and underscores deleterious late mitotic events, including the uncoupling of anaphase A and B and cytokinesis. Elevation of CDK2 to a level similar to CDK1 overcomes the mitotic defects caused by CDK1 deficiency, indicating that the relatively low concentration of CDK2 accounts for the defective anaphase. Collectively, these results reveal that the difference between G2-M and G1-S CDKs in human cells is essentially quantitative.

Disrupting microtubule dynamics with spindle poisons activates the spindle-assembly checkpoint (SAC) and induces mitotic cell death. However, mitotic exit can occur prematurely without proper chromosomal segregation or cytokinesis by a process termed mitotic slippage. It remains controversial whether mitotic slippage increases the cytotoxicity of spindle poisons or the converse. Altering the SAC induces either mitotic cell death or mitotic slippage. While knockout of MAD2-binding protein p31comet strengthened the SAC and promoted mitotic cell death, knockout of TRIP13 had the opposite effect of triggering mitotic slippage. We demonstrated that mitotic slippage prevented mitotic cell death caused by spindle poisons, but reduced subsequent long-term survival. Weakening of the SAC also reduced cell survival in response to spindle perturbation insufficient for triggering mitotic slippage, of which mitotic exit was characterized by displaced chromosomes during metaphase. In either mitotic slippage or mitotic exit with missegregated chromosomes, cell death occurred only after one cell cycle following mitotic exit and increased progressively during subsequent cell cycles. Consistent with these results, transient inhibition of the SAC using an MPS1 inhibitor acted synergistically with spindle perturbation in inducing chromosome missegregation and cytotoxicity. The specific temporal patterns of cell death after mitotic exit with weakened SAC may reconcile the contradictory results from many previous studies.

It is well established that Aurora kinases perform critical functions during mitosis. It has become increasingly clear that the Aurora kinases also perform a myriad of non-mitotic functions including DNA damage response. The available evidence indicates that inhibition Aurora kinase A (AURKA) may contribute to the G2 DNA damage checkpoint through AURKA's functions in PLK1 and CDC25B activation. Both AURKA and Aurora kinase B (AURKB) are also essential in mitotic DNA damage response that guard against DNA damage-induced chromosome segregation errors, including the control of abscission checkpoint and prevention of micronuclei formation. Dysregulation of Aurora kinases can trigger DNA damage in mitosis that is sensed in the subsequent G1 by a p53-dependent postmitotic checkpoint. Aurora kinases are themselves linked to the G1 DNA damage checkpoint through p53 and p73 pathways. Finally, several lines of evidence provide a connection between Aurora kinases and DNA repair and apoptotic pathways. Although more studies are required to provide a comprehensive picture of how cells respond to DNA damage, these findings indicate that both AURKA and AURKB are inextricably linked to pathways guarding against DNA damage. They also provide a rationale to support more detailed studies on the synergism between small-molecule inhibitors against Aurora kinases and DNA-damaging agents in cancer therapies.

Characterizing the functions of essential cell cycle control genes requires tight and rapid inducible gene inactivation. Drawbacks of current conditional depletion approaches include slow responses and incomplete depletion. We demonstrated that by integrating the tetracycline-controlled promoter system and the auxin-inducible degron (AID) system together, AID-tagged proteins can be downregulated more efficiently than the individual technology alone. When used in conjunction with CRISPR-Cas9-mediated disruption of the endogenous locus, this system facilitates the analysis of essential genes by allowing rapid and tight conditional depletion, as we have demonstrated using several cell cycle-regulatory genes including cyclin A, CDK2, and TRIP13. The vectors constructed in this study allow expression of AID-fusion proteins under the control of tetracycline-controlled promoters and should be useful in studies requiring rapid and tight suppression of gene expression in mammalian cells.

Shugoshin (SGO1) plays a pivotal role in sister chromatid cohesion during mitosis by protecting the centromeric cohesin from mitotic kinases and WAPL. Mammalian cells contain at least 6 alternatively spliced isoforms of SGO1. The relationship between the canonical SGO1A with shorter isoforms including SGO1C remains obscure. Here we show that SGO1C was unable to replace the loss of SGO1A. Instead, expression of SGO1C alone induced aberrant mitosis similar to depletion of SGO1A, promoting premature sister chromatid separation, activation of the spindle-assembly checkpoint, and mitotic arrest. In disagreement with previously published data, we found that SGO1C localized to kinetochores. However, the ability to induce aberrant mitosis did not correlate with its kinetochore localization. SGO1C mutants that abolished binding to kinetochores still triggered premature sister chromatid separation. We provide evidence that SGO1C-mediated mitotic arrest involved the sequestering of PP2A–B56 pool. Accordingly, SGO1C mutants that abolished binding to PP2A localized to kinetochores but did not induce aberrant mitosis. These studies imply that the expression of SGO1C should be tightly regulated to prevent dominant-negative effects on SGO1A and genome instability.

// Joyce P.Y. Mak 1 , Wing Yu Man 1 , Jeremy P.H. Chow 1 , Hoi Tang Ma 1 , Randy Y.C. Poon 1 1 Division of Life Science, Center for Cancer Research, and State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong Correspondence to: Randy Y.C. Poon, e-mail: rycpoon@ust.hk Keywords: DNA damage checkpoint, mitosis, mitotic catastrophe, nasopharyngeal carcinoma, WEE1 Received: March 02, 2015 Accepted: May 09, 2015 Published: May 21, 2015 ABSTRACT Nasopharyngeal carcinoma (NPC) is a rare but highly invasive cancer. As radiotherapy is the primary treatment for NPC, this offers a rationale to investigate if uncoupling the DNA damage responses can sensitize this cancer type. The G 2 DNA damage checkpoint is controlled by a cascade of protein kinases: ATM/ATR, which phosphorylates CHK1/CHK2, which in turn phosphorylates WEE1. A number of small molecule inhibitors have been developed against these kinases as potential therapeutic agents. Here we demonstrated that compare to that in immortalized nasopharyngeal epithelial cells, ATR, CHK1, and WEE1 were overexpressed in NPC cell lines. Inhibitors of these kinases were unable to promote extensive mitotic catastrophe in ionizing radiation-treated NPC cells, indicating that they are not very effective radiosensitizer for this cancer. In the absence of prior irradiation, however, mitotic catastrophe could be induced with inhibitors against CHK1 (AZD7762) or WEE1 (MK-1775). NPC cells were more sensitive to WEE1 inactivation than nasopharyngeal epithelial cells. Targeting CHK1 and WEE1 together induced more extensive mitotic catastrophe than the individual components alone. Taken together, our results show that NPC cells depend on CHK1 and WEE1 activity for growth and that inhibitors of these kinases may serve as potential therapeutics for NPC.

Mitosis is choreographed by a number of protein kinases including polo-like kinases and Aurora kinases. As these kinases are frequently dysregulated in cancers, small-molecule inhibitors have been developed for targeted anticancer therapies. Given that PLK1 and Aurora kinases possess both unique functions as well as co-regulate multiple mitotic events, whether pharmacological inhibition of these kinases together can enhance mitotic catastrophe remains an outstanding issue to be determined. Using concentrations of inhibitors that did not induce severe mitotic defects on their own, we found that both the metaphase arrest and mitotic slippage induced by inhibitors targeting Aurora A and Aurora B (MK-5108 and Barasertib respectively) were enhanced by a PLK1 inhibitor (BI 2536). We found that PLK1 is overexpressed in cells from nasopharyngeal carcinoma, a highly invasive cancer with poor prognosis, in comparison to normal nasopharyngeal epithelial cells. Nasopharyngeal carcinoma cells were more sensitive to BI 2536 as a single agent and co-inhibition with Aurora kinases than normal cells. These observations underscore the mechanism and potential benefits of targeting PLK1 and Aurora kinases to induce mitotic catastrophe in cancer cells.

Summary Mutations of DISC1 (disrupted-in-schizophrenia 1) have been associated with major psychiatric disorders. Despite the hundreds of DISC1-binding proteins reported, almost nothing is known about how DISC1 interacts with other proteins structurally to impact human brain development. Here we solved the high-resolution structure of DISC1 C-terminal tail in complex with its binding domain of Ndel1. Mechanistically, DISC1 regulates Ndel1's kinetochore attachment, but not its centrosome localization, during mitosis. Functionally, disrupting DISC1/Ndel1 complex formation prolongs mitotic length and interferes with cell-cycle progression in human cells, and it causes cell-cycle deficits of radial glial cells in the embryonic mouse cortex and human forebrain organoids. We also observed similar deficits in organoids derived from schizophrenia patient induced pluripotent stem cells (iPSCs) with a DISC1 mutation that disrupts its interaction with Ndel1. Our study uncovers a new mechanism of action for DISC1 based on its structure, and it has implications for how genetic insults may contribute to psychiatric disorders.

// Joyce P.Y. Mak 1 , Wing Yu Man 1 , Hoi Tang Ma 1 and Randy Y.C. Poon 1 1 Division of Life Science, Center for Cancer Research, and State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong Correspondence: Randy Y.C. Poon, email: // Keywords : anticancer drugs; checkpoint; DNA damage; mitosis; mitotic catastrophe Received : June 14, 2014 Accepted : September 24, 2014 Published : September 25, 2014 Abstract The ATR–CHK1–WEE1 kinase cascade’s functions in the DNA damage checkpoints are well established. Moreover, its roles in the unperturbed cell cycle are also increasingly being recognized. In this connection, a number of small-molecule inhibitors of ATR, CHK1, and WEE1 are being evaluated in clinical trials. Understanding precisely how cells respond to different concentrations of inhibitors is therefore of paramount importance and has broad clinical implications. Here we present evidence that in the absence of DNA damage, pharmacological inactivation of ATR was less effective in inducing mitotic catastrophe than inhibition of WEE1 and CHK1. Small-molecule inhibitors of CHK1 (AZD7762) or WEE1 (MK-1775) induced mitotic catastrophe, as characterized by dephosphorylation of CDK1 Tyr15 , phosphorylation of histone H3 Ser10 , and apoptosis. Unexpectedly, partial inhibition of WEE1 and CHK1 had the opposite effect of accelerating the cell cycle without inducing apoptosis, thereby increasing the overall cell proliferation. This was also corroborated by the finding that cell proliferation was enhanced by kinase-inactive versions of WEE1. We demonstrated that these potential limitations of the inhibitors could be overcome by targeting more than one components of the ATR–CHK1–WEE1 simultaneously. These observations reveal insights into the complex responses to pharmacological inactivation of the ATR–CHK1–WEE1 axis.

Nasopharyngeal carcinoma is a rare but highly invasive cancer. As options of agents for effective combination chemoradiotherapy for advanced nasopharyngeal carcinoma are limited, novel therapeutic approaches are desperately needed. The ubiquitin ligase CHFR is known to target PARP1 for degradation and is epigenetically inactivated in nasopharyngeal carcinoma. We present evidence that PARP1 protein is indeed overexpressed in nasopharyngeal carcinoma cells in comparison with immortalized normal nasopharyngeal epithelial cells. Tissue microarray analysis also indicated that PARP1 protein is significantly elevated in primary nasopharyngeal carcinoma tissues, with strong correlation with all stages of nasopharyngeal carcinoma development. We found that the PARP inhibitor AZD2281 (olaparib) increased DNA damage, cell-cycle arrest, and apoptosis in nasopharyngeal carcinoma cells challenged with ionizing radiation or temozolomide. Isobologram analysis confirmed that the cytotoxicity triggered by AZD2281 and DNA-damaging agents was synergistic. Finally, AZD2281 also enhanced the tumor-inhibitory effects of ionizing radiation in animal xenograft models. These observations implicate that PARP1 overexpression is an early event in nasopharyngeal carcinoma development and provide a molecular basis of using PARP inhibitors to potentiate treatment of nasopharyngeal carcinoma with radio- and chemotherapy. Mol Cancer Ther; 12(11); 2517–28. ©2013 AACR.

// Wei Xia 1 , Chung Mau Lo 1 , Randy Y.C. Poon 2 , Tan To Cheung 1 , Albert C.Y. Chan 1 , Lin Chen 1 , Sitian Yang 1 , George S.W. Tsao 3 and Xiao Qi Wang 1, 4 1 Department of Surgery, The University of Hong Kong, Hong Kong, China 2 Division of Life Science, Center for Cancer Research, and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China 3 School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China 4 State Key Laboratory for Liver Research, The University of Hong Kong, Hong Kong, China Correspondence to: Xiao Qi Wang, email: xqwang@hku.hk George S.W. Tsao, email: gswtsao@hku.hk Keywords: cyclin D1, Smad2/3/Smad4, cancer stem cells, Smad inhibitor, chemosensitization Received: November 02, 2016 Accepted: March 09, 2017 Published: March 21, 2017 ABSTRACT Understanding cancer stem cell (CSC) maintenance pathways is critical for the development of CSC-targeting therapy. Here, we investigated the functional role of the cyclin D1-dependent activation of Smad2/3 and Smad4 in hepatocellular carcinoma (HCC) CSCs and in HCC primary tumors. Cyclin D1 sphere-derived xenograft tumor models were employed to evaluate the therapeutic effects of a Smad inhibitor in combination with chemotherapy. Cyclin D1 overexpression confers stemness properties by enhancing single sphere formation, enhancing the CD90+ and EpCAM+ population, increasing stemness gene expression, and increasing chemoresistance. Cyclin D1 interacts with and activates Smad2/3 and Smad4 to result in cyclin D1-Smad2/3-Smad4 signaling-regulated liver CSC self-renewal. The cyclin D1-dependent activation of Smad2/3 and Smad4 is also found in HCC patients and predicts disease progression. A Smad inhibitor impaired cyclin D1-Smad-mediated self-renewal, resulting in the chemosensitization. Thus, pretreatment with a Smad inhibitor followed by chemotherapy not only successfully suppressed tumor growth but also eliminated 57% of the tumors in a cyclin D1 sphere-derived xenograft model. Together, The cyclin D1-mediated activation of Smad2/3 and Smad4 is an important regulatory mechanism in liver CSC self-renewal and stemness. Accordingly, a Smad inhibitor induced CSC differentiation and consequently significant chemosensitization, which could be an effective strategy to target CSCs.

// Shan Huang 1 , Rui Tang 1 , Randy Y.C. Poon 1 1 Division of Life Science, Center for Cancer Research, and State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong Correspondence to: Randy Y.C. Poon, email: rycpoon@ust.hk Keywords: apoptosis, BCL-2, microtubule, mitosis Abbreviations: doxycycline, Dox; nocodazole, NOC; paclitaxel, PTX Received: March 07, 2016 Accepted: April 28, 2016 Published: May 25, 2016 ABSTRACT Microtubule inhibitors including taxanes and vinca alkaloids are among the most widely used anticancer agents. Disrupting the microtubules activates the spindle-assembly checkpoint and traps cells in mitosis. Whether cells subsequently undergo mitotic cell death is an important factor for the effectiveness of the anticancer agents. Given that apoptosis accounts for the majority of mitotic cell death induced by microtubule inhibitors, we performed a systematic study to determine which members of the anti-apoptotic BCL-2 family are involved in determining the duration of mitotic block before cell death or slippage. Depletion of several anti-apoptotic BCL-2-like proteins significantly shortened the time before apoptosis. Among these proteins, BCL-W has not been previously characterized to play a role in mitotic cell death. Although the expression of BCL-W remained constant during mitotic block, it varied significantly between different cell lines. Knockdown of BCL-W with siRNA or disruption of the BCL-W gene with CRISPR-Cas9 speeded up mitotic cell death. Conversely, overexpression of BCL-W delayed mitotic cell death, extending the mitotic block to allow mitotic slippage. Taken together, these results showed that BCL-W contributes to the threshold of anti-apoptotic activity during mitosis.

Antimitotic spindle poisons are among the most important chemotherapeutic agents available. However, precocious mitotic exit by mitotic slippage limits the cytotoxicity of spindle poisons. The MAD2-binding protein p31(comet) is implicated in silencing the spindle assembly checkpoint after all kinetochores are attached to spindles. In this study, we report that the levels of p31(comet) and MAD2 in different cell lines are closely linked with susceptibility to mitotic slippage. Down-regulation of p31(comet) increased the sensitivity of multiple cancer cell lines to spindle poisons, including nocodazole, vincristine, and Taxol. In the absence of p31(comet), lower concentrations of spindle poisons were required to induce mitotic block. The delay in checkpoint silencing was induced by an accumulation of mitotic checkpoint complexes. The increase in the duration of mitotic block after p31(comet) depletion resulted in a dramatic increase in mitotic cell death upon challenge with spindle poisons. Significantly, cells that are normally prone to mitotic slippage and resistant to spindle disruption-mediated mitotic death were also sensitized after p31(comet) depletion. These results highlight the importance of p31(comet) in checkpoint silencing and its potential as a target for antimitotic therapies.

The current paradigm states that exit from mitosis is triggered by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) acting in concert with an activator called CDC20. While this has been well established for a number of systems, the evidence of a critical role of CDC20 in somatic cells is not unequivocal. In this study, we reexamined whether mitotic exit can occur properly after CDC20 is depleted. Using single-cell analysis, we found that CDC20 depletion with small interfering RNAs (siRNAs) significantly impaired the degradation of APC/C substrates and delayed mitotic exit in various cancer cell lines. The recruitment of cyclin B1 to the core APC/C was defective after CDC20 downregulation. Nevertheless, CDC20-depleted cells were still able to complete mitosis, albeit requiring twice the normal time. Intriguingly, a high level of cyclin-dependent kinase 1 (CDK1)-inhibitory phosphorylation was induced during mitotic exit in CDC20-depleted cells. The expression of an siRNA-resistant CDC20 rescued both the mitotic exit delay and the CDK1-inhibitory phosphorylation. Moreover, the expression of a nonphosphorylatable CDK1 mutant or the downregulation of WEE1 and MYT1 abolished mitotic exit in CDC20-depleted cells. These findings indicate that, in the absence of sufficient APC/C activity, an alternative mechanism that utilized the classic inhibitory phosphorylation of CDK1 could mediate mitotic exit.

Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule–kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.

金沢大学医薬保健研究域薬学系, Cdc25 dual specificity phosphatases positively regulate the cell cycle by activating cyclin-dependent kinase/cyclin complexes. Of the three mammalian Cdc25 isoforms, Cdc25A is phosphorylated by genotoxic stress-activated Chk1 or Chk2, which triggers its SCFβ-TrCP-mediated degradation. However, the roles of Cdc25B and Cdc25C in cell stress checkpoints remain inconclusive. We herein report that c-Jun NH2-terminal kinase (JNK) induces the degradation of Cdc25B. Nongenotoxic stress induced by anisomycin caused rapid degradation of Cdc25B as well as Cdc25A. Cdc25B degradation was dependent mainly on JNK and partially on p38 mitogen-activated protein kinase (p38). Accordingly, cotransfection with JNK1, JNK2, or p38 destabilized Cdc25B. In vitro kinase assays and site-directed mutagenesis experiments revealed that the critical JNK and p38 phosphorylation site in Cdc25B was Ser101. Cdc25B with Ser101 mutated to alanine was refractory to anisomycin-induced degradation, and cells expressing such mutant Cdc25B proteins were able to override the anisomycin-induced G2 arrest. These results highlight the importance of a novel JNK/p38-Cdc25B axis for a nongenotoxic stress-induced cell cycle checkpoint. ©2009 American Association for Cancer Research.

For mouse embryonic stem (ES) cells, the importance of the Sand G 2 cell cycle checkpoints for genomic integrity is increased by the absence of the G 1 checkpoint. We have investigated ionizing radiation (IR)-mediated cell cycle checkpoints in undifferentiated and retinoic acid-differentiated human embryonal carcinoma (EC) cells. Like mouse ES cells, human EC cells did not undergo G 1 arrest after IR but displayed a prominent S-phase delay followed by a G 2-phase delay. In contrast, although differentiated EC cells also failed to arrest at G 1-phase after IR, they quickly exited S-phase and arrested in G 2-phase. In differentiated EC cells, the G 2-M-phase cyclin B1/CDC2 complex was upregulated after IR, but the G 1-S-phase cyclin E and the cyclin E/ CDK2 complex were expressed at constitutively low levels, which could be an important factor distinguishing DNA damage responses between undifferentiated and differentiated EC cells. S-phase arrest and expression of p21 could be inhibited by 7-hydroxystaurosporine, suggesting that the ataxia-telangiectasia and Rad-3-related-checkpoint kinase 1 (ATR-CHK1), and p21 pathways might play a role in the IR-mediated S-phase checkpoint in EC cells. IR-mediated phosphorylation of ataxia-telangiectasia mutated, (CHK1), and checkpoint kinase 2 were distinctly higher in undiffer- entiated EC cells compared with differentiated EC cells. Combined with the prominent S and G 2 checkpoints and a more efficient DNA damage repair system, these mechanisms operate together in the maintenance of genome stability for EC cells. ©AlphaMed Press., published_or_final_version

Although cells can exit mitotic block aberrantly by mitotic slippage, they are prevented from becoming tetraploids by a p53-dependent postmitotic checkpoint. Intriguingly, disruption of the spindle-assembly checkpoint also compromises the postmitotic checkpoint. The precise mechanism of the interplay between these two pivotal checkpoints is not known. We found that after prolonged nocodazole exposure, the postmitotic checkpoint was facilitated by p53. We demonstrated that although disruption of the mitotic block by a MAD2-binding protein promoted slippage, it did not influence the activation of p53. Both p53 and its downstream target p21CIP1/WAF1 were activated at the same rate irrespective of whether the spindle-assembly checkpoint was enforced or not. The accelerated S phase entry, as reflected by the premature accumulation of cyclin E relative to the activation of p21CIP1/WAF1, is the reason for the uncoupling of the postmitotic checkpoint. In support of this hypothesis, forced premature mitotic exit with a specific CDK1 inhibitor triggered DNA replication without affecting the kinetics of p53 activation. Finally, replication after checkpoint bypass was boosted by elevating the level of cyclin E. These observations indicate that disruption of the spindle-assembly checkpoint does not directly influence p53 activation, but the shortening of the mitotic arrest allows cyclin E-CDK2 to be activated before the accumulation of p21CIP1/WAF1. These data underscore the critical relationship between the spindle-assembly checkpoint and the postmitotic checkpoint in safeguarding chromosomal stability.

The G2 DNA damage checkpoint is one of the most important mechanisms controlling G2–mitosis transition. The kinase Greatwall (MASTL in human) promotes normal G2–mitosis transition by inhibiting PP2A via ARPP19 and ENSA. In this study, we demonstrate that MASTL is critical for maintaining genome integrity after DNA damage. Although MASTL did not affect the activation of DNA damage responses and subsequent repair, it determined the timing of entry into mitosis and the subsequent fate of the recovering cells. Constitutively active MASTL promoted dephosphorylation of CDK1Tyr15 and accelerated mitotic entry after DNA damage. Conversely, downregulation of MASTL or ARPP19/ENSA delayed mitotic entry. Remarkably, APC/C was activated precociously, resulting in the damaged cells progressing from G2 directly to G1 and skipping mitosis all together. Collectively, these results established that precise control of MASTL is essential to couple DNA damage to mitosis through the rate of mitotic entry and APC/C activation.

The mechanisms of how signaling pathways are coordinated and integrated for the maintenance of the self-renewal of human embryonic stem cells (hESCs) and the acquisition of pluripotency in reprogramming are still only partly understood. CDK1 is a key regulator of mitosis. Recently, CDK1 has been shown to be involved in regulating self-renewal of stem cells, even though the mechanistic role of how CDK1 regulates pluripotency is unknown. In this report, we aim to understand how CDK1 can control pluripotency by reducing CDK1 activity to a level that has no effect on cell cycle progression. We demonstrated that high levels of CDK1 is associated with the pluripotency stage of hESCs; and decreased CDK1 activity to a level without perturbing the cell cycle is sufficient to induce differentiation. CDK1 specifically targets the phosphorylation of PDK1 and consequently the activity of PI3K/Akt and its effectors ERK and GSK3β. Evidence of the reversion of inactive CDK1-mediated differentiation by the inhibition of Akt signaling effectors suggests that the CDK1-PDK1-PI3K/Akt kinase cascade is a functional signaling pathway for the pluripotency of hESCs. Moreover, cyclin B1-CDK1 complexes promote somatic reprogramming efficiency, probably by regulating the maturation of induced pluripotent stem cells (iPSCs), as cyclin B1 stimulates a higher cellular level of LIN28A, suggesting that monitoring iPSC factors could be a new path for the enhancement of reprogramming efficiency. Together, we demonstrate an essential role for the CDK1-PDK1-PI3K/Akt kinase signaling pathway in the regulation of self-renewal, differentiation, and somatic reprogramming, which provides a novel kinase cascade mechanism for pluripotency control and acquisition.

The multidomain-containing cellular protein BS69 interacts with adenovirus E1A and several other viral and cellular factors, and acts as a transcription repressor. Here, we show that BS69 is involved in the p53-p21Cip1-mediated senescence pathway. Knockdown of BS69 by RNA interference in human primary fibroblasts results in elevated levels of p21Cip1 and the appearance of several senescent markers, including enhanced senescence-associated beta-galactosidase activity and formation of senescence-associated heterochromatin foci. Importantly, knockdown of either p53 or p21Cip1, but not p16(INK4a) or Rb, allows cells to bypass premature senescence that is induced by BS69 knockdown. Furthermore, we show that BS69 forms complexes with both p53 and p400, and that BS69 associates with the p21Cip1 promoter through p53. Together, our data indicate that BS69 is involved in cellular senescence mainly through the p53-p21Cip1 pathway.

Summary: Biochemical studies have indicated that p31comet and TRIP13 are critical for inactivating MAD2. To address unequivocally whether p31comet and TRIP13 are required for mitotic exit at the cellular level, their genes were ablated either individually or together in human cells. Neither p31comet nor TRIP13 were absolutely required for unperturbed mitosis. MAD2 inactivation was only partially impaired in p31comet-deficient cells. In contrast, TRIP13-deficient cells contained MAD2 exclusively in the C-MAD2 conformation. Our results indicate that although p31comet enhanced TRIP13-mediated MAD2 conversion, it was not absolutely necessary for the process. Paradoxically, TRIP13-deficient cells were unable to activate the spindle-assembly checkpoint, revealing that cells lacking the ability to inactivate MAD2 were incapable in mounting a checkpoint response. These results establish a paradigm of the roles of p31comet and TRIP13 in both checkpoint activation and inactivation. : Mitotic exit requires the inactivation of MAD2. Ma and Poon show that MAD2 inactivation in mammalian cells requires TRIP13 and is stimulated by p31comet. Moreover, TRIP13-deficient cells containing only C-MAD2 are unable to activate the spindle-assembly checkpoint. Keywords: MAD2, mitosis, mitotic exit, p31comet, spindle-assembly checkpoint

Polyploidization occurs during normal development as well as during tumorigenesis. In this study, we investigated if the responses to genotoxic stress in cancer cells are influenced by the ploidy. Prolonged treatment of Hep3B cells with the spindle inhibitor nocodazole resulted in mitotic slippage, followed by re-replication of the DNA to produce polyploids. Reintroduction of p53 restored the checkpoints and suppressed polyploidization. Remarkably, a stable tetraploidy cell line could be generated from Hep3B by a transient nocodazole treatment followed by a period of recovery. Using this novel tetraploid system, we found that tetraploidization increased the cell volume without significantly affecting the cell cycle. Although tetraploidization was accompanied by an increase in centrosome number, the majority of mitoses in the tetraploid cells remained bipolar. Polyploidization sensitized cells to genotoxic stress inflicted by ionizing radiation and topoisomerase inhibitors without affecting the sensitivity to spindle inhibitors. Accordingly, more gamma-H2AX foci were induced by radiation in tetraploids than in normal Hep3B cells. Likewise, primary tetraploid human fibroblasts displayed higher gamma-H2AX foci formation than diploid human fibroblasts. An implication for chemotherapy is that some cancer cells can be sensitized to genotoxic agents by a preceding step that induces polyploidization.

The tumor suppressor p53 is negatively regulated by the ubiquitin ligase MDM2. The MDM2 recognition site is at the NH2-terminal region of p53, but the positions of the actual ubiquitination acceptor sites are less well defined. Lysine residues at the COOH-terminal region of p53 are implicated as sites for ubiquitination and other post-translational modifications. Unexpectedly, we found that substitution of the COOH-terminal lysine residues did not diminish MDM2-mediated ubiquitination. Ubiquitination was not abolished even after the entire COOH-terminal regulatory region was removed. Using a method involving in vitro proteolytic cleavage at specific sites after ubiquitination, we found that p53 was ubiquitinated at the NH2-terminal portion of the protein. The lysine residue within the transactivation domain is probably not essential for ubiquitination, as substitution with an arginine did not affect MDM2 binding or ubiquitination. In contrast, several conserved lysine residues in the DNA-binding domain are critical for p53 ubiquitination. Removal of the DNA-binding domain reduced ubiquitination and increased the stability of p53. These data provide evidence that in addition to the COOH-terminal residues, p53 may also be ubiquitinated at sites in the DNA-binding domain. (Mol Cancer Res 2006;4(1)15–25)

Cyclin A is destroyed during mitosis by the ubiquitin-proteasome system. Like cyclin B, a destruction box (D-box) motif is required for the destruction of cyclin A. However, cyclin A degradation is more complicated than cyclin B because cyclin A's D-box motif is more extensive and proteolysis involves complex signaling in some organisms. In this study, we found that in addition to the D-box, the region between residues 123-157 also contributed to the ubiquitination and degradation of human cyclin A. Indeed, removal of the bulk of the N-terminal regulatory domain was needed to completely stabilize cyclin A and eliminate ubiquitination. A putative second RxxL motif around residue 138 played only a minor role in cyclin A degradation. To distinguish between sequences recognized by the ubiquitination machinery and the ubiquitin acceptor sites per se, we utilized a novel approach involving in vitro cleavage of cyclin A after ubiquitination. We found that several lysine residues proximal to the D-box (Lys37, Lys54, and Lys68) were ubiquitin acceptor sites. Cyclin A lacking the three lysine residues was degraded slower than the wild-type protein. Although these lysines were normally used, ubiquitination could shift to other cryptic sites when the preferred sites were unavailable, suggesting the exact positions of the ubiquitin chains also contributed to degradation. Together, these data revealed that ubiquitination does not occur randomly on cyclin A and open up questions on the precise function of the D-box.

Cyclins are discovered as proteins that accumulate progressively through interphase and disappear abruptly at mitosis during each cell cycle. In mammalian cells, cyclin A accumulates from late G1 phase and is destroyed before metaphase, and cyclin B is destroyed slightly later at anaphase. The abundance of the mitotic cyclins is mainly regulated at the levels of transcription and proteolysis. Transcription is stimulated and repressed by several transcription factors, including B-MYB, E2F, FOXM1, and NF-Y. Elements in the promoter, including CCRE/CDE and CHR, are in part responsible for the cell cycle oscillation of transcription. Destruction of the mitotic cyclins is carried out by the ubiquitin ligases APC/C(CDC20) and APC/C(CDH1). Central to our knowledge is the understanding of how APC/C is turned on from anaphase to early G1 phase, and turned off from late G1 till the spindle-assembly checkpoint is deactivated in metaphase. Reciprocal actions of cyclin-dependent kinases (CDKs) on APC/C, as well as on the SCF complexes ensure that the mitotic cyclins are destroyed only at the proper time.

Topoisomerase II poisons like Adriamycin (ADR, doxorubicin) are clinically important chemotherapeutic agents. Adriamycin-induced DNA damage checkpoint activates ATM and ATR, which could in turn inhibit the cell cycle engine through either CHK1 or CHK2. In this study, we characterized whether CHK1 or CHK2 is required for Adriamycin-induced checkpoint. We found that both CHK1 and CHK2 were phosphorylated after Adriamycin treatment. Several lines of evidence from dominant-negative mutants, short hairpin RNA (shRNA), and knockout cells indicated that CHK1, but not CHK2, is critical for Adriamycin-induced cell cycle arrest. Disruption of CHK1 function bypassed the checkpoint, as manifested by the increase in CDC25A, activation of CDC2, increase in histone H3 phosphorylation, and reduction in cell survival after Adriamycin treatment. In contrast, CHK2 is dispensable for Adriamycin-induced responses. Finally, we found that CHK1 was upregulated in primary hepatocellular carcinoma (HCC), albeit as an inactive form. The presence of a stockpile of dormant CHK1 in cancer cells may have important implications for treatments like topoisomerase II poisons. Collectively, the available data underscore the pivotal role of CHK1 in checkpoint responses to a variety of stresses.

Most cancer cells are aneuploid. The chromosomal instability hypothesis contends that aneuploidy is the catalyst for transformation, whereas the gene mutation hypothesis asserts that cancer is driven by mutations to proto-oncogenes and tumor-suppressor genes, with the aneuploidy a side effect of tumorigenesis. Because genotoxic stress induced by “culture shock” can obscure the transforming potential of exogenous genes, we cultured wild-type and p53-/- mouse embryo fibroblasts in a more physiological (serum-free) environment. Under these conditions, the cells were immortal and, more importantly, chromosomally stable. Expression of oncogenic H-RasV12 did not induce senescence, but sensitized these cells to p53-dependent apoptosis. In addition, H-RasV12 induced chromosomal instability, as well as accumulation and phosphorylation of p53. Significantly, whereas cells grown under standard conditions could be transformed by coexpression of H-RasV12 and E1A, the chromosomally stable cells were refractory to transformation, as measured by anchorage-independent growth and tumor formation in nude mice. These oncogenes required a third genetic alteration that abolished the p53 pathway to create a permissive environment that promotes rapid chromosomal instability and transformation. Oncogene-induced chromosomal instability and transformation was attenuated by antioxidants. These data indicate that chromosomal instability could be a catalyst for oncogenic transformation, and bring together aspects of the chromosomal instability hypothesis and the gene mutation hypothesis for tumorigenesis.

The tumor suppressor p53 is transcription factor composed of four identical subunits. The majority of the mutations in p53 are missense mutations that impair DNA binding. On the other hand, the p53-related p63 and p73 genes are rarely mutated, but many cell types express natural variants lacking the N-terminal transactivation domain (NDelta). Compelling evidence indicates that both the DNA binding-defective and NDelta mutants can impair the function of wild-type p53 in a dominant-negative manner. Interestingly, it is uncertain how many mutant subunit(s) a p53 tetramer can tolerate. In this study, we first made theoretical predictions based on the number of mutant p53 monomers needed to inactivate a tetramer and then tested how well the experimental data fit the predicted values. Surprisingly, these experiments reveal that DNA binding-defective p53 mutants (R249S and R273H) are very ineffective in impairing the transcriptional activity of p53: at least three mutants are required to inactivate a tetramer. In marked contrast, p53NDelta is a very potent inhibitor of p53: one NDelta subunit per tetramer is sufficient to abolish the transcriptional activity. DNA binding is not necessary for the NDelta proteins to inactivate p53. Similarly, NDelta variants of p63 and p73 are also powerful inhibitors of members of the p53 family. These results have important implications for our thinking about the mechanism of tumorigenesis involving missense p53 mutants or the N-terminally truncated isoforms.

CHK1 and CHK2 are key mediators that link the machineries that monitor DNA integrity to components of the cell cycle engine. Despite the similarity and potential redundancy in their functions, CHK1 and CHK2 are unrelated protein kinases, each having a distinctive regulatory domain. Here we compare how the regulatory domains of human CHK1 and CHK2 modulate the respective kinase activities. Recombinant CHK1 has only low basal activity when expressed in cultured cells. Surprisingly, disruption of the C-terminal regulatory domain activates CHK1 even in the absence of stress. Unlike the full-length protein, C-terminally truncated CHK1 displays autophosphorylation, phosphorylates CDC25C on Ser(216), and delays cell cycle progression. Intriguingly, enzymatic activity decreases when the entire regulatory domain is removed, suggesting that the regulatory domain contains both inhibitory and stimulatory elements. Conversely, the kinase domain suppresses Ser(345) phosphorylation, a major ATM/ATR phosphorylation site in the regulatory domain. In marked contrast, CHK2 expressed in either mammalian cells or in bacteria is already active as a kinase against itself and CDC25C and can delay cell cycle progression. Unlike CHK1, disruption of the regulatory domain of CHK2 abolishes its kinase activity. Moreover, the regulatory domain of CHK2, but not that of CHK1, can oligomerize. Finally, CHK1 but not CHK2 is phosphorylated during the spindle assembly checkpoint, which correlates with the inhibition of the kinase. The mitotic phosphorylation of CHK1 requires the regulatory domain, does not involve Ser(345), and is independent on ATM. Collectively, these data reveal the very different mode of regulation between CHK1 and CHK2.

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G1but seemed to retract to a G2-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G1entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.

Mammalian DNA replication is an elegantly choreographed process in which multiple components are assembled at the origins to form the prereplication complex. Formation and activation of the prereplication complex requires coordinate actions of G1 and S phase cyclin-dependent kinases. Cyclin E-CDK2 and cyclin A-CDK2, together with DBF4-CDC7, phosphorylate several components of the prereplication complex and replication machinery. In this review, we summarize the current understanding of the mechanism of initiation of DNA replication in mammalian cells. The roles of cyclin A/E-CDK2 complexes in driving replication, their relationship with other regulators of S phase, and their role in keeping replication to only once per cell cycle will be discussed. In addition, an important issue is the checks and balances that prevent inappropriate DNA replication, and how a breakdown in these checkpoints can lead to genomic instability and cancer. A critical mediator of these checkpoints, ATM, signals through a comprehe...

Cyclin A is particularly interesting among the cyclin family because it can activate two different cyclin-dependent kinases (CDKs) and functions in both S phase and mitosis. An embryonic form of cyclin A that is only essential for spermatogenesis is also present in some organisms. In S phase, phosphorylation of components of the DNA replication machinery such as CDC6 by cyclin A-CDK is believed to be important for initiation of DNA replication and to restrict the initiation to only once per cell cycle. In mitosis, the precise role of cyclin A is still obscure, but it may contribute to the control of cyclin B stability. Cyclin A starts to accumulate during S phase and is abruptly destroyed before metaphase. The synthesis of cyclin A is mainly controlled at the transcription level, involving E2F and other transcription factors. Removal of cyclin A is carried out by ubiquitin-mediated proteolysis, but whether the same anaphase-promoting complex/cyclosome targeting subunits are used as for cyclin B is debatable. Consistent with its role as a key cell cycle regulator, expression of cyclin A is found to be elevated in a variety of tumors.

Background: The cyclin-dependent kinases (CDKs) CDC2 and CDK2 are key regulators of the cell cycle. The expression of the CDK alone does not necessary reflect their true activities because they are highly regulated by post-translational mechanisms. Human hepatocellular carcinoma (HCC) is one of the most common cancers in the world, but the kinase activities of CDKs in HCC have not been examined. Methods: Here we examined the protein expression and kinase activities associated with CDC2 and CDK2 in HCC and the corresponding non-tumorous liver tissues. Results: CDC2 and CDK2 are activated in HCC in over 70% and 80% of the cases, respectively, but have little correlation with clinical parameters and PCNA expression. Interestingly, PCNA was readily detectable in extracts from non-tumorous liver, but more than 60% of samples contain higher concentration of PCNA in HCC than the corresponding non-tumorous tissues. CDC2 and CDK2 are generally activated in the same HCC samples, but the extent of their activation varied significantly, suggesting that the pathways leading to the activation of CDC2 and CDK2 can be regulated independently. Both positive regulators of CDK activity like cyclins and CDKs, and negative regulators of CDK activity like p21CIP1/WAF1 and Thr14/Tyr15 phosphorylation were up-regulated in HCC. Conclusion: CDC2 and CDK2 are activated in HCC, and this may be due to a complex interplay between the level of the cyclin, CDK, CDK inhibitors, and inhibitory phosphorylation.

DNA double-stranded breaks (DSBs) are highly deleterious and need to be repaired for maintaining genomic stability and viability. Cells resolve DSBs via non-homologous end-joining or homologous recombination repair (HRR). The simultaneous downregulation of both repair mechanisms induces synthetic lethality. Accordingly, PARP1 inhibitors (PARPi) are being used to target cancers with defective HRR such as cancers with BRCA1/2 mutations. Similar to BRCA1/2, ATM is involved in HRR; and ATM deficiency enhances sensitivity towards PARPi. But unlike BRCA1/2, ATM can be targeted using various small-molecule inhibitors (ATMi) in cancers containing wild type ATM. Using nasopharyngeal carcinoma cell lines as models, we found that the combined treatment of PARPi and ATMi led to the accumulation of DSBs, activation of the G2 DNA damage checkpoint, reduced mitotic entry, and diminished cell proliferation. Co-inhibition of ATM and PARP1 induced robust DNA damage responses without triggering mitotic catastrophe. In agreement with this, PARPi induced more DNA damage in ATM-knockout cells than wild type cells. Conversely, ATMi promoted more DNA damage when PARP1 was ablated. Although it is believed that PARPi exerts its effects by both inhibiting PARP1 catalytic activities and trapping PARP1 at DNA breaks, we found that the synergism between ATMi and PARPi did not require PARP1 trapping. These results provide insights on developing combined treatment of PARPi and ATMi into anti-cancer therapies for cancers without HRR or NHEJ deficiencies. Citation Format: Joyce Pui Ying Mak, Randy Yat Choi Poon. Synthetic lethality induced by pharmacological inhibition of ATM and PARP1 [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A20.

Cyclins and cyclin-dependent kinases (CDKs) are key regulators of the human cell cycle. Here we have directly measured the concentrations of the G(1) and G(2) cyclins and their CDK partners in highly synchronized human cervical carcinoma cells (HeLa). To determine the exact concentrations of cyclins and CDKs in the cell extracts, we developed a relatively simple method that combined the use of (35)S-labeled standards produced in rabbit reticulocyte lysates and immunoblotting with specific antibodies. Using this approach, we formally demonstrated that CDC2 and CDK2 are in excess of their cyclin partners. We found that the concentrations of cyclin A2 and cyclin B1 (at their peak levels in the G(2) phase) were about 30-fold less than that of their partner CDC2. The peak levels of cyclin A2 and cyclin E1, at the G(2) phase and G(1) phase, respectively, were only about 8-fold less than that of their partner CDK2. These ratios are in good agreement with size fractionation analysis of the relative amount of monomeric and complexed forms of CDC2 and CDK2 in the cell. All the cyclin A2 and cyclin E1 are in complexes with CDC2 and CDK2, but there are some indications that a significant portion of cyclin B1 may not be in complex with CDC2. Furthermore, we also demonstrated that the concentration of the CDK inhibitor p21(CIP1/WAF1) induced after DNA damage is sufficient to overcome the cyclin-CDK2 complexes in MCF-7 cells. These direct quantitations formally confirmed the long-held presumption that CDKs are in excess of the cyclins in the cell. Moreover, similar approaches can be used to measure the concentration of any protein in cell-free extracts.

Many cyclins are degraded by the ubiquitination/proteasome pathways involving the anaphase-promoting complex and SCF complexes. These degradations are frequently dependent on phosphorylation by cyclin-dependent kinases (CDKs), providing a self-limiting mechanism for CDK activity. Here we present evidence from in vitro and in vivo assay systems that the degradation of human cyclin A can be inhibited by kinase-inactive mutants of CDK2 and CDC2. One obvious interpretation of these results is that like other cyclins, CDK-dependent phosphorylation of the cyclin A may be involved in cyclin A degradation. Our data indicated that CDK2 can phosphorylate cyclin A on Ser-154. Site-directed mutagenesis of Ser-154 abolished the phosphorylation by recombinant CDK2 in vitro and the majority of cyclin A phosphorylation in the cell. Activation of CDK2 and binding to SKP2 or p27(KIP1) were not affected by the phosphorylation of Ser-154. Surprising, in marked contrast to cyclin E, where phosphorylation of Thr-380 by CDK2 is required for proteolysis, degradation of cyclin A was not affected by Ser-154 phosphorylation. It is likely that the stabilization of cyclin A by the kinase-inactive CDKs was mainly due to a cell cycle effect. These data suggest an important difference between the regulation of cyclin A and cyclin E.

During differentiation, skeletal muscle cells withdraw from the cell cycle and fuse into multinucleated myotubes. Unlike quiescent cells, however, these cells cannot be induced to reenter S phase by means of growth factor stimulation. The studies reported here document that both the retinoblastoma protein (Rb) and the cyclin-dependent kinase (cdk) inhibitor p21 contribute to this unresponsiveness. We show that the inactivation of Rb and p21 through the binding of the adenovirus E1A protein leads to the induction of DNA replication in differentiated muscle cells. Moreover, inactivation of p21 by E1A results in the restoration of cyclin E–cdk2 activity, a kinase made nonfunctional by the binding of p21 and whose protein levels in differentiated muscle cells is relatively low in amount. We also show that restoration of kinase activity leads to the phosphorylation of Rb but that this in itself is not sufficient for allowing differentiated muscle cells to reenter the cell cycle. All the results obtained are consistent with the fact that Rb is functioning downstream of p21 and that the activities of these two proteins may be linked in sustaining the postmitotic state.

In tissue culture systems, p21 and p27 inhibit cyclin-dependent kinase (CDK) activity and cell cycle progression in response to numerous stimuli, but little is known about their involvement in cell growth in vivo. We examined the modulation of CDK activity by these proteins after 70% partial hepatectomy (PH), an in vivo model of synchronous hepatocyte cell cycle progression. After PH in BALB/c mice, p21 was induced during the prereplicative (G1) phase and was maximally expressed after peak hepatocyte DNA synthesis. p27 was present in quiescent liver and was minimally induced after PH. p21 and p27 immunoprecipitated with CDK2, CDK4, and cyclin D1 in the regenerating liver. The activity of CDK2-, CDK4- and cyclin D1-associated kinases was upregulated after PH, and maximal activity of these enzyme complexes corresponded to peak DNA synthesis. Immunodepletion experiments suggested that p27 plays a role in downregulating CDK2 activity before and after peak DNA synthesis. Compared to cogenic wild-type mice, p21-/- mice demonstrated evidence of markedly accelerated hepatocyte progression through G1 phase after PH: DNA synthesis, upregulation of cyclin A and PCNA, induction of cyclin D1- and CDK2-associated kinase activity, and appearance of a phosphorylated retinoblastoma protein (Rb) species occurred earlier in the p21-/- mice. These results suggest that p21 and p27 modulate CDK activity in the regenerating liver, and that p21 regulates the rate of progression through G1 phase of the cell cycle in vivo.

The mitotic kinesin KIF11 (also called Eg5) plays critical roles in spindle functions. Although a number of small-molecule inhibitors of KIF11 are currently in clinical development, drug-resistance could be developed through compensation by another kinesin called KIF15. Using a newly developed infrared-based cell system, we discovered that the effectiveness of one of the latest generations of KIF11 inhibitor (SB743921) could be enhanced with several inhibitors of Aurora A kinase. Evidence including live-cell imaging and isobologram analysis indicated that targeting KIF11 and Aurora A together promoted monoastral spindle formation and mitotic catastrophe synergistically, supporting a model of parallel pathways of centrosome regulation by Aurora A and KIF11. We also developed a KIF15-dependent SB743921-resistance cell model. Significantly, the drug-resistance could also be overcome with Aurora A inhibitors. These results provide a molecular basis for increasing the effectiveness of Aurora A and KIF11 inhibitors and tackling problems of drug resistance.

We report the label-free enumeration of human colorectal-carcinoma cells from blood lymphocytes by using interdigitated ring-array microelectrodes; this enumeration was based on the dielectrophoretic selection of cells. Because of the novel design of the device, a continuous flow of cells is uniformly distributed into parallel streams through 300 rings (~40 μm in diameter each) that are integrated into the electrode digits. Using this array, 82% of cancer cells were recovered and 99% of blood lymphocytes were removed. Most of the cancer cells recovered were viable (94%) and could be cultivated for8 days, during which period they retained their normal cell morphology and proliferation rates. The recovery rate correlated closely with cancer-cell loadings in spiked samples and this relationship was linear over a range of at least 2 orders of magnitude. Importantly, because of the 3D structure of the rings, these results were obtained at a high cell-loading concentration (10(7)cells/mL). The rings could be further optimized for use in accurate label-free identification and measurement of circulating tumor cells in cancer research and disease management.

The activity of most protein kinases is highly regulated, typically via phosphorylation and/or subunit association. However, the folding of protein kinases into an active state or a form capable of activation is now emerging as another important step through which they can be regulated. The 50-kDa protein Cdc37 and the associated heat-shock protein Hsp90 have been found to bind to, and be required for the activity of, diverse protein kinases, including Cdk4, v-Src, Raf and SEVENLESS. Together, Cdc37 and Hsp90 may act as a general chaperone for protein kinases, in particular those involved in signal-transduction pathways and cell-cycle control.

Sister chromatid separation depends on the activity of separase, which in turn requires the proteolysis of its inhibitor, securin. It has been speculated that securin also supports the activation of separase. In this study, we found that PTTG1 was the major securin isoform expressed in most normal and cancer cell lines. Remarkably, a highly homologous isoform called PTTG2 was unable to interact with separase. Using chimeras between PTTG1 and PTTG2 and other approaches, we pinpointed a single amino acid that accounted for the loss of securin function in PTTG2. Mutation of the homologous position in PTTG1 (H(134)) switched PTTG1 from an inhibitor into an activator of separase. In agreement with this, PTTG1 lacking H(134) was able to trigger premature sister chromatid separation. Conversely, introduction of H(134) into PTTG2 is sufficient to allow it to bind separase. These data demonstrate that while the motif containing H(134) has a strong affinity for separase and is involved in inhibiting it, another domain(s) is involved in activating separase and has a weaker affinity for it. Although PTTG2 lacks securin function, its differences from PTTG1 provide evidence of independent inhibitory and activating functions of PTTG1 on separase.

The cyclin-dependent kinase (CDK) inhibitor p21 is induced by the tumor suppressor gene product p53 and is thought to be important for the arrest of the cell cycle following DNA damage. Here we have investigated the contribution of p21 in inhibiting different cyclin-CDK complexes that drive different cell cycle transitions following UV irradiation-induced DNA damage in normal human fibroblasts and immortalized rodent fibroblasts. When cells were exposed to a low dose of UV irradiation, both p53 and p21 were induced; the protein kinase activities associated with Cdc2, Cdk2, and Cdk4 were inhibited; and there was a good correlation between their inhibition and binding to p21. p21 alone is likely to be sufficient for the inhibition of Cdk2 because all the cyclin-complexed forms of Cdk2 were associated with p21 after irradiation. In contrast, only a small proportion of Cdk4 and Cdc2 was complexed with p21, although the level of Cdk4 associated with either p21 or p27 was increased after irradiation. Furthermore, recombinant p21 added to an unirradiated cell lysate at the same level as that induced by irradiation damage inhibited only the kinase activity associated with Cdk2. Cdc2 is likely to be inhibited by Thr-14/Tyr-15 phosphorylation after irradiation because Cdc2 was tyrosine-phosphorylated, and recombinant Cdc25 was able to increase its kinase activity significantly. Taken together, these results suggest that different CDKs are inhibited by different mechanisms following UV-induced DNA damage: Cdk2 is inhibited by the elevated level of p21; Cdk4 is inhibited by cooperation of p21 with other CDK inhibitors, like p27, and possibly by phosphorylation; and Cdc2 is inhibited by Thr-14/Tyr-15 phosphorylation. It is likely that these underlying mechanisms that inactivate CDKs are similar for other kinds of DNA damage.

Cancer cells have been characterized with activated mutant oncogenes and inactivated or deleted tumor suppressor genes. Cancer cells are also aneuploid, displaying a jumble of chromosomal anomalies including gain or loss of whole chromosomes or transposed chromosomal fragments. Whether mutation of specific genes or aneuploidy is more critical for tumorigenesis is very much a contentious issue. We recently showed that activated oncogenes induce oxidative damage that is exacerbated by conventional cell culture conditions. This "culture shock" or a loss of p53 function creates a precarious environment that permits oncogenes to induce rapid chromosomal instability and transformation. We found that mutant genes and aneuploidy were prerequisites and collaborators for neoplastic transformation.

A number of small-molecule inhibitors of Aurora kinases have been developed and are undergoing clinical trials for anti-cancer therapies. Different Aurora kinases, however, behave as very different targets: while inhibition of Aurora A (AURKA) induces a delay in mitotic exit, inhibition of Aurora B (AURKB) triggers mitotic slippage. Furthermore, while it is evident that p53 is regulated by Aurora kinase-dependent phosphorylation, how p53 may in turn regulate Aurora kinases remains mysterious. To address these issues, isogenic p53-containing and -negative cells were exposed to classic inhibitors that target both AURKA and AURKB (Alisertib and ZM447439), as well as to new generation of inhibitors that target AURKA (MK-5108), AURKB (Barasertib) individually. The fate of individual cells was then tracked with time-lapse microscopy. Remarkably, loss of p53, either by gene disruption or small interfering RNA-mediated depletion, sensitized cells to inhibition of both AURKA and AURKB, promoting mitotic arrest and slippage respectively. As the p53-dependent post-mitotic checkpoint is also important for preventing genome reduplication after mitotic slippage, these studies indicate that the loss of p53 in cancer cells represents a major opportunity for anti-cancer drugs targeting the Aurora kinases.